Among gene products that relate to transplantation antigens are the products of the Human Leukocyte Antigen (HLA) complex, also known as the major histocompatibility complex (MHC), located on the short arm of chromosome 6. The HLA antigens are divided into two classes depending on their structure. The genetic loci denoted HLA-A -B, and -C code for the HLA Class I antigens, and HLA-DP, -DQ and -DR code for the HLA Class II antigens.
HLA Class II molecules are composed of two non-covalently linked glycoproteins, the a chain and the highly polymorphic .beta. chain. Each chain contains one extracellular domain, a transmembrane segment and a cytoplasmic tail. The structure of the .alpha. and .beta. chains and their genes have been elucidated. All known Class II genes are similar in structure and encoded by exons 1-4, with exon 5 coding for an untranslated region. The DP, DQ and DR loci all consist of multiple genes. A total of twelve class II genes have been identified. In some haplotypes, some class II genes do not code for a functional peptide and are classified as pseudogenes.
Regulation of HLA class II antigen expression occurs in part through a series of promoter regions such as the J, W, X (including X.sub.1 and X.sub.2), and Y boxes, and the gamma interferon response element. The X (including X.sub.1 and X.sub.2) and Y boxes are known to be required in the transcriptional regulation of all class II promoters. Ono, S. J. et al., Proc. Natl. Acad. Sci. (USA) (1991) 88: 4304-4308.
Transcription of HLA-DR.alpha. Class II can be activated by RF-X (Regulatory Factors-X) which binds to the X-box region (-110 to -95) of the DR.alpha. promoter. RF-X and its binding site, the X-box are unique and have a high specificity for each other. The DNA binding domain of RF-X consists of 91 amino acids with a basic stretch and shares no notable homology with other known DNA binding motifs (Reith et al. (1990), Genes Dev., Vol. 4(9), pp. 1528-40). RF-X binds only the X-box; substitutions in the X-box are generally not well tolerated by RF-X. (Hasegawa et al. (1991), Nucleic Acids Res., Vol 19(6), pp. 1243-49). The X-box sequence is an atypical promoter site, being neither palindromic nor dyad symmetric. Additionally no other sequence (using the program Eugene) shows exact homology with the X-box. The X-box is conserved in humans. No other known cloned transcription factors bind to this entire region of the X-box in the same manner.
HLA antigens are implicated in the survival of cell grafts or transplants. Although there is acceptable graft survival in the first year for nearly all types of transplants, by five and ten years after transplantation only 40-50% of all grafts are still functioning. This low rate is due to the relentless attack of the immune system on the graft. In addition, death rates of 1-5% are recorded even at the best transplant centers. Drugs are commonly used to control immune responses and prevent graft rejection, and death is often an indirect result of this drug administration.
The drugs used to control immune responses usually cause a non-specific depression of the immune system. A patient with a depressed immune system is far more susceptible to develop life-threatening infections and a variety of neoplasia. The low rate of long term success, and serious risks of infection and cancer are the two main challenges now facing the entire field of tissue and organ transplantation.
It has been suggested that graft rejection can be prevented or reduced by reducing the levels of exposed HLA antigens on the surface of transplant cells. Faustman, D. et al., Science (1991) 252:1700-1702, observed that xenograft survival was increased by masking HLA class I surface antigens with F(ab').sub.2 antibody fragments to HLA class I or tissue specific epitopes.
One way to reduce the level of cell surface transplantation antigens is to retard (downregulate) the expression of the transplantation antigen genes.
Generally, eucaryotic gene expression may be regulated at any of the steps from DNA transcription to RNA translation to protein; and it is generally agreed that gene expression is at the level of transcription. In order for transcription to occur, transcription factors must bind distinct regulatory sites or promoters on the gene. Once bound, transcription factors may interact with RNA polymerase or other factors to activate or repress transcription. Some transcription factors are constitutively expressed in specific cells while others may be transiently activated in response to various physiological signals (such as cAMP, IFN-.gamma., etc.). Thus in a given cell transcription of particular genes depends on which transcription factors are present in that cell type and/or whether the signals to activate the transcription factors are present.
Agents such as actinomycin (an intercalator) have been used to block transcription in a nonspecific manner. A variety of approaches to sequence-specific gene modulation include use of antisense oligonucleotides and antigene oligonucleotides (triple helix formers). These are limited in general or in particular instances. Antisense oligonucleotides block gene expression by targeting mRNA while triple helix forming oligos target double-stranded DNA. Inaccessibility of the target mRNA due to RNA secondary structure can limit the usefulness of antisense methods; triple helix approaches are limited by poor nuclear access, chromatin structure (bypassing histones), and the need for targeting a homopurine-homopyrimidine stretch. Degradation of oligonucleotides by exonucleases an excessive binding to untargeted cellular factors can limit the effectiveness of both antisense and triple helix methods. Chemical modification of oligos can improve nuclease resistance but can also result in increased toxicity, reduced binding affinity, and lower activity (Cook 1991, Crooke 1991).
J. T. Holt (1991), Antisense Res. Dev., Vol. 1(4), pp. 65-9, has shown that by providing excess DNA binding sites, specific transcription factors can be quenched and are thereby prevented from binding to endogenous DNA.
Chu et al. (1991) Nucl. Acids Res. 19: 6958, describe DNA structures in "hairpin" and "dumbbell" configurations containing CRE and TRE sequences, and reported using them in vitro as substitutes for regular double-stranded DNA to bind CREB and JUN, respectively, in gel shift assays.
Chu et al. (1992) Nucl. Acids Res. 21: 5857-5858, demonstrate that in nuclear extracts, dumbbell DNA is much more stable than double-stranded or hairpin DNA. However, the nicking of dumbbell DNA in human serum, caused by endonuclease degradation of the single-stranded loops, is a potential problem, since it converts dumbbell DNA to a double-stranded form that is no more stable in the nucleus than standard double-stranded DNA molecules.
International patent publication WO92/19732 describes "closed" oligonucleotides that can be used as "sense" or "antisense" molecules, with the advantage of being resistant to exonucleases. Among the variety of "closed" structures are "dumbbell" configurations in which the ends are closed by virtue of addition links of thymidine nucleotides. Use of the closed "sense" oligonucleotides to bind protein factors having as affinity for RNA or DNA sequences or structures is suggested.
Single-strand circular DNA where a portion becomes double-stranded have been used as an experimental system for studying local thermal stability in DNA (Wemmer et al. (1985), Nucl. Acid. Res., Vol. 13(23), pp. 8611-21); as models for hairpins and cruciforms (Erie et al. (1989), Biochemistry, Vol. 28(1), pp. 268-73); and as models for comparison to nicked or gapped DNA (Snowden-Ifft et al. (1990), Biochemistry, Vol. 29(25), pp. 6017-25).